This invention relates generally to scanning optical microscopes with a confocal capability.
A confocal optical microscope is one in which a single spot of illumination is focused on to a specimen and a detector of light receives light derived from that spot only. An image is built up progressively by scanning the spot over the specimen or by scanning the specimen relative to a stationary spot Minsky (U.S. Pat. No. 3,013,467) first described such a microscope, including the use of a pinhole aperture and optical means by which the light from the specimen was focused on that aperture. This and other early apparatus suffered from the difficulty that the pinhole aperture was required to be very small for optimum confocal operation. For example, Wilson and Carlini, in an article published in the Journal of Microscopy 149, pp 51-66 (1988) calculated that for a commonly-used type of microscope objective lens, the optimum pinhole diameter was 10 xcexcm. This made the manufacture and correct alignment of confocal microscopes difficult. White (U.S. Pat. No. 5,032,720) taught the use of a large extra optical magnification in such a microscope, allowing the use of an aperture of several millimeters in diameter without compromise of the confocal function of the microscope. In the apparatus of White, the extra magnification was achieved by using the microscope eyepiece to project a real image through a very large distance (greater than 1 meter). It is well known to those skilled in the art of optics that magnification can also be achieved by means of a telescope. Fukuyama (U.S. Pat. No. 5,225,671) described two types of telescope for this purpose, namely a Galilean telescope consisting of a positive and a negative lens and a catadioptric telescope consisting of convex and concave reflecting elements. In a brochure published by Bio-Rad Ltd., a model of confocal scan head (microRadiance) is shown with a telescope consisting of two positive lenses.
It is desirable to be able optionally to remove the extra magnification, making the scan head non-confocal. This is required, for example, to increase the signal strength at the detector, or to decrease the optical-sectioning property of the microscope to facilitate searching in three dimensions for a specimen feature. Another important reason is to collect more signals in a multiphoton epifluorescence microscope than can be collected with confocal geometry. The desirability of the latter is discussed by Denk, Piston and Webb in The Handbook of Biological Confocal Microscopy edited by J. Pawley, pp 445-458 Plenum Press (1995). A disadvantage of the telescope-based systems described above is that the lenses or mirrors of the telescope must be positioned very precisely, so it is difficult to remove the telescope, substitute optics giving a lower magnification and then replace the telescope in a sufficiently reproducible way. For example, the telescope used in a system manufactured by Bio-Rad Ltd. has a power of approximately 15 times. The lateral tolerance in projecting the spot on to the detector iris is 0.2 mm at 160 mm distance, which corresponds to 1.2 mrad of angle. The mechanical tolerance in angular position of the telescope axis is 1.2/15 or 0.08 mrad, which is difficult to maintain by ordinary mechanical mountings.
The present application concerns a scanning optical microscope with means by which the magnification at the detector diaphragm can be varied by the introduction of an additional optical unit which can be inserted or removed without disturbing the telescope, thus allowing rapid and accurate switching to and from confocal function.
The invention can be viewed as an improvement over the disclosure of U.S. Pat. No. 5,334,830.
According to the invention, there is provided a scanning optical microscope having a telescope and a light detector with a detector aperture through which in use light from a specimen is focussed on the detector, in combination with an attachment comprising a unit having optical power insertable into the light path through the microscope in order to alter the magnification of the image of the specimen in the plane of the detector aperture, wherein the microscope is a confocal microscope without the unit inserted in the light path and a non-confocal microscope with the unit inserted in the light path, characterised in that the unit is insertable into the light path at a location between the telescope and the light detector.
The present invention thus provides a scanning optical microscope with an attachment unit which can be inserted reversibly into and out of the light path of a confocal microscope leading to the detector which, when inserted, modifies the magnification of the real image of the object at the level of the detector aperture or iris. Preferably, the unit consists of lens or reflector assemblies of positive and negative power which cause the magnification of the image in the plane of the detector iris to be changed. The unit is used in addition to the telescope and keeps the apparent position of the exit pupil of the telescope constant. If, during confocal imaging, a collector lens is used between the detector and the confocal iris to image the exit pupil of the telescope on the detector, the collector lens preferably continues to function in this way even when the unit has been inserted and special features of the design of the unit can make this possible. The method of use of this unit varies according to the magnification the unit produces and mode of imaging. A form providing a negative magnification (i.e. reducing the magnification of the telescope) can be used preferably with the detector iris fully open, to maximise signal collection in multiphoton imaging and to provide high-sensitivity epifluorescence imaging without confocal performance. A form providing positive magnification can be used with a restricted aperture to improve confocal performance or to allow the use of special non-standard aperture masks.